Project Summary The proposed studies will determine in molecular detail the mechanism(s) by which poly(A)-binding protein Pab1 phase-separates to form hydrogel assemblies during stress. Cellular stresses cause the evolutionarily conserved, putatively adaptive formation stress granules. Pab1 is a defining marker of stress granules, and Pab1's phase separation precedes stress granule formation. Dysregulation of phase separation of multiple RNA-binding proteins is linked to pathological protein aggregation associated with major neurodegenerative disorders. The mechanism(s) of phase separation is an area of active inquiry. The key barrier has been a lack of tractable in vitro models of biologically relevant phase separation by an RNA-binding protein. We have broken through this barrier. In published studies (Wallace et al., Cell 2015; Riback et al., Cell 2017), we have established that Pab1 phase-separates into hydrogel droplets in response to physiological stress conditions, and that interfering with hydrogel droplet formation disrupts budding yeast's ability to survive thermal and starvation stress. No previously described system combines a stress-triggered phase separation process, phenotypic consequences during stress, and the ability to reconstitute phase separation at physiological concentrations and conditions. Pab1/poly(A)-binding protein is conserved across eukaryotes and is a core marker of stress granules, making it a promising model for determining the molecular basis of stress-induced phase separation. We will study Pab1's phase separation at multiple scales, including the influence of RNA, using a combination in vitro and in vivo approaches to determine how this unique phase separation process is encoded. We will test specific hypotheses, such as the involvement of electrostatic interactions between RNA-binding domains and hydrophobic interactions between IDRs, using mutational approaches combined with hydrogen exchange mass spectrometry (HX-MS), NMR, dynamic light scattering, and small-angle X-ray scattering. We with further develop a mesoscale assay which provides rich information about how Pab1 interactions influence the properties of the resulting assemblies (viscosity, nucleation versus growth, changes in phase) thought to contribute to its biological functions. The project requires the expertise of two complementary investigators. Drummond brings his extensive experience in Pab1 and stress biology while Sosnick brings his expertise in protein folding and chemistry.